RNA-DNA Hybrids Support Recombination-Based Telomere Maintenance in Fission Yeast.

A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant "alternative lengthening of telomeres" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood.

FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops.

Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells.

TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.

The shelterin protein TRF2 assembles protective T loops at chromosome ends by stimulating intramolecular invasion of the telomeric G-rich single-stranded DNA (ssDNA) overhang into the duplex telomeric array. The other shelterin factor, TRF1, is thought to mainly facilitate telomeric dsDNA replication without directly participating in end protection. Here we show that in vitro human TRF2 stimulates invasion of G-rich TERRA-like RNA into telomeric dsDNA, leading to formation of telomeric RNA-DNA hybrids (telR loops).

Human cactin interacts with DHX8 and SRRM2 to assure efficient pre-mRNA splicing and sister chromatid cohesion.

Cactins constitute a family of eukaryotic proteins broadly conserved from yeast to human and required for fundamental processes such as cell proliferation, genome stability maintenance, organismal development and immune response. Cactin proteins have been found to associate with the spliceosome in several model organisms, nevertheless their molecular functions await elucidation. Here we show that depletion of human cactin leads to premature sister chromatid separation, genome instability and cell proliferation arrest.

TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.

Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation.

Eukaryotic Ribosome Assembly and Nuclear Export.

Accurate translation of the genetic code into functional polypeptides is key to cellular growth and proliferation. This essential process is carried out by the ribosome, a ribonucleoprotein complex of remarkable size and intricacy. Although the structure of the mature ribosome has provided insight into the mechanism of translation, our knowledge regarding the assembly, quality control, and intracellular targeting of this molecular machine is still emerging.

Human Mpn1 promotes post-transcriptional processing and stability of U6atac.

Mpn1 is an exoribonuclease that modifies the spliceosomal small nuclear RNA (snRNA) U6 by trimming its oligouridine tail and introducing a cyclic phosphate group (>p). Mpn1 deficiency induces U6 3' end misprocessing, accelerated U6 decay and pre-mRNA splicing defects. Mutations in the human MPN1 gene are associated with the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN).

Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels.

The telomeric transcriptome comprises multiple long non-coding RNAs generated by transcription of linear chromosome ends. In a screening performed in Schizosaccharomyces pombe, we identified factors modulating the cellular levels of the telomeric transcriptome. Among these factors, Cay1 is the fission yeast member of the conserved family of Cactins, uncharacterized proteins crucial for cell growth and survival.

Dissecting ribosome assembly and transport in budding yeast.

Construction of the eukaryotic ribosome begins in the nucleolus and requires >300 evolutionarily conserved nonribosomal trans-acting factors, which transiently associate with preribosomal subunits at distinct assembly stages. A subset of trans-acting and transport factors passage assembled preribosomal subunits in a functionally inactive state through the nuclear pore complexes (NPC) into the cytoplasm, where they undergo final maturation before initiating translation. Here, we summarize the repertoire of tools developed in the model organism budding yeast that are spearheading the functional analyses of trans-acting factors involved in the assembly and intracellular transport of preribosomal subunits.

Assembly and nuclear export of pre-ribosomal particles in budding yeast.

The ribosome is responsible for the final step of decoding genetic information into proteins. Therefore, correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps.

NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2).

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2.

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116.

The Mpn1 RNA exonuclease: cellular functions and implication in disease.

Recent studies from independent laboratories have decisively disclosed the identity of the long-sought 3-5' RNA exonuclease that trims posttranscriptionally the oligouridine tail of U6, which is the small catalytic non-coding RNA promoting premRNA splicing within the spliceosome. This exonuclease, dubbed Mpn1 or Usb1, is a highly conserved enzyme that specifically removes uridines from the 3' end of U6, and directly generates terminal 2',3' cyclic phosphate groups. Mutations in the human gene encoding hMpn1 have been reported in patients diagnosed with the rare genodermatosis Clericuzio-type poikiloderma with neutropenia (PN).

Role of Mex67-Mtr2 in the nuclear export of 40S pre-ribosomes.

Nuclear export of mRNAs and pre-ribosomal subunits (pre40S and pre60S) is fundamental to all eukaryotes. While genetic approaches in budding yeast have identified bona fide export factors for mRNAs and pre60S subunits, little is known regarding nuclear export of pre40S subunits. The yeast heterodimeric transport receptor Mex67-Mtr2 (TAP-p15 in humans) binds mRNAs and pre60S subunits in the nucleus and facilitates their passage through the nuclear pore complex (NPC) into the cytoplasm by interacting with Phe-Gly (FG)-rich nucleoporins that line its transport channel.

The telomeric transcriptome: from fission yeast to mammals.

The ends of linear eukaryotic chromosomes are transcribed into different species of non-coding transcripts (the telomeric transcriptome), including TERRA (telomeric repeat-containing RNA) molecules; however, the functions associated with the telomeric transcriptome remain elusive. Experimental evidence accumulated during the past few years indicates that the transcriptional activity of telomeres is changed in cells in which the integrity of the telomeres or the heterochromatic state of chromosome ends is altered. On the contrary transcription of a telomere appears not to be influenced by its length.

UPF1: a leader at the end of chromosomes.

The human helicase and ATPase up-frameshift suppressor 1 (UPF1), traditionally known as a major player in several RNA quality control mechanisms, is emerging as a crucial caretaker of the stability of the genome. Work from my laboratory has provided insight into the function of UPF1 during DNA metabolism and has revealed that this versatile enzyme sustains the proper replication of telomeres, the protective structures located at the ends of linear eukaryotic chromosomes. We have supplied direct evidence that telomere replication is not completed in cells with compromised UPF1 function, leading to the accumulation of DNA damage and telomere abnormalities.

Getting ready to translate: cytoplasmic maturation of eukaryotic ribosomes.

The ribosome is the 'universal ribozyme' that is responsible for the final step of decoding genetic information into proteins. While the function of the ribosome is being elucidated at the atomic level, in comparison, little is known regarding its assembly in vivo and intracellular transport. In contrast to prokaryotic ribosomes, the construction of eukaryotic ribosomes, which begins in the nucleolus, requires >200 evolutionary conserved non-ribosomal trans-acting factors, which transiently associate with pre-ribosomal subunits at distinct assembly stages and perform specific maturation steps.

Sem1: a versatile "molecular glue"?

The evolutionary conserved protein Sem1/Dss1 is a bona fide subunit of the regulatory particle (RP) of the proteasome and in mammalian cells stabilizes the tumor suppressor protein BRCA2. A recent study from our laboratory has revealed an unexpected non- proteasomal role of Sem1 in mRNA export. We found that Sem1, independent of the RP, is a functional component of the nuclear pore associated TREX-2 complex that is directly involved in the dynamic relocalization of a subset of DNA loci to the nuclear periphery.

Maturation of eukaryotic ribosomes: acquisition of functionality.

In eukaryotic cells, ribosomes are pre-assembled in the nucleus and exported to the cytoplasm where they undergo final maturation. This involves the release of trans-acting shuttling factors, transport factors, incorporation of the remaining ribosomal proteins, and final rRNA processing steps. Recent work, particularly on the large (60S) ribosomal subunit, has confirmed that the 60S subunit is exported from the nucleus in a functionally inactive state.

The telomeric transcriptome and SMG proteins at the crossroads.

Telomeres constitute the heterochromatic structures at the ends of linear eukaryotic chromosomes and are essential for the maintenance of genome stability. The longstanding belief that telomeres are transcriptionally silent has recently been overturned by the discovery of a telomeric transcriptome comprising telomeric repeat-containing RNA (TERRA) molecules. In mammalian cells, TERRA remains associated with telomeres after its transcription, and this association is regulated by SMG proteins, renowned for their involvement in several RNA metabolism pathways.

Telomeres: the silence is broken.

The ends of linear eukaryotic chromosomes, telomeres, distinguish natural chromosome ends from DNA double stranded breaks and thus promote genome stability. Telomeres comprise a repetitive DNA skeleton, which is wrapped in specific protein complexes. Recent data indicate that an additional building block of telomeres is RNA and that the longstanding idea that telomeres are silent genomic regions needs to be overturned.

Adjunct duties for karyopherins: regulating septin sumoylation.

Karyopherins are shuttling transport receptors regulated by the small GTPase Ran, which move cargo between the nucleus and cytoplasm by passing through the nuclear pore complexes. A recent paper in Journal of Cell Biology (Makhnevych et al., 2007) highlights an additional role for karyopherins during mitosis, in regulating the sumoylation status of the septin rings.